Reaction mass of Amines, N-tallow alkyltrimethylenedi-, mono(2-ethylhexanoates), Amines, N-tallow alkyltrimethylenedi-, acetates and n-tallow-1,3-diaminopropane ditallate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2017-04-24 to 2017-06-26 with the definitive exposure phase from 2017-06-20 to 2017-06-23,

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
All loading levels and the controls were analytically verified via LC-MS/MS at the start (0 hours) and at the end of the exposure (72 hours) with algae. Additionally, analysis was done on the stock solution at test start. To evaluate if spiking the test solutions is performed correctly and to evaluate the stability of the test item under test conditions, separate replicates without algae of the two loadings 0.0320 and 0.0800 mg/L were analysed at test start and test end. Adsorption to glass was analytically verified from all concentration levels and the two additional loadings without algae at the end of the exposure. The vessels were rinsed carefully before analysis, to remove adhering algae. The analyzed constituents are considered representative for the exposure to the diamine fraction of the product. The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).

Vehicle:

no

Details on test solutions:

Water Accommodated Fraction (WAF)
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000) is to expose the organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted solvents. Using this approach, aqueous media were prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. After completion of mixing and following a settlement period, the test item phase was separated by siphon and the test organisms were exposed to the aqueous phase, the WAF (which may contain dissolved and/or suspended and/or emulsified fractions of the test item mixture). Exposure was expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of the test item in the WAF.

Preparation of the water accommodated fractio
Five water accommodated fractions (WAF) were prepared with an nominal loading of the test item of 0.00513 – 0.0128 – 0.0320 – 0.0800 – 0.200 mg/L (spacing factor 2.5). The loading levels are based on the results of a preliminary range finding test (non-GLP, open system) and the first definitive test.

For a loading of 0.5 mg/L an appropriate amount of the test item was weighed out. The test item was applied onto a glass slide. The glass slide with the test item was inserted in a brown glass flask with an appropriate amount of dilution water (see Table 2). The dispersion was shaken for at least 24 hours with 20 rpm at room temperature. After a separation phase of 1 hour, WAF was removed by siphoning (from the approximate centre of the glass flask). The resulting water accommodated fraction (WAF) was used as a stock solution. All test loading levels were prepared by diluting the WAF with a loading of 0.5 mg/L.

Test loading
Per definition of the WAF, all terms related to concentration level
were given as loading level because partly dissolved compounds and mixtures cannot be related to concentrations.

Control
Six replicates (without test item) were tested under the same test conditions as the test item replicates.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK
- Strain: SAG 61.81
- Source (laboratory, culture collection): SAG, Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux corresponding to 35 - 70 µE ∙ m-2 ∙ s-1 for 24 h per day.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

Not specified

Test temperature:

min.: 22.0 max.: 23.5 mean value: 22.8

pH:

Nominal test Mean measured pH-values
item loadings initial main
constituents concentration
[mg/L] [mg m.c./L] Start; 0 hours End; 72 hours
0.200 0.0330 7.52 8.03
0.0800 0.0132 7.56 8.34
0.0320 0.00529 7.62 8.94
0.0128 0.00211 7.72 9.10
0.00513 0.000846 7.87 9.20
Control 7.90 9.36

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

Nominal test substance concentrations: 0, 0.00512, 0.0128, 0.032, 0.08 and 0.2 mg/L
Nominal amine fraction concentrations: 0, 0.00267, 0.00668, 0.0167, 0.04176 and 0.1044 mg/L (=52.2% of test substance).
Nominal main constituent concentrations: 0, 0.00235, 0.00587, 0.0147, 0.0367 and 0.0917 mg/L (main constituents C18:1 diamine, C16 diamine and C18 diamine represent 87.8% of amine fraction in the test item)
Measured main constituent concentrations: 0, 0.000846, 0.00211, 0.00529, 0.0132 and 0.0330 mg/L (Geometric mean measured concentrations of the main constituents)
Calculated concentration of the amine fraction in the test item: 0, 0.00964, 0.00240, 0.00603, 0.0150 and 0.0376 mg/L (calculated from geometric mean by multiplying by 1/0.878)
Calculated concentration of the test item: 0, 0.00185, 0.00460, 0.01154, 0.0288 and 0.072 mg/L (calculated from geometric mean of the amine fraction and multiplying by 1/0.522)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 2 - 5 x 10^3 cells/mL, Actual: mean 6839 cells/mL
- Control end cells density: Mean 2025631 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Composition of Dilution Water
according to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2 x 6 H2O 12
CaCl2 x 2 H2O 18
MgSO4 x 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064
Na2EDTA x 2 H2O 0.1
H3BO3 0.185
MnCl2 x 4 H2O 0.415
ZnCl2 3 x 10^-3
Na2MoO4 x 2 H2O 7 x 10^-3
CoCl2 x 6 H2O 1.5 x 10^-3
CuCl2 x 2 H2O 1 x 10^-5
NaHCO3 50
pH 8.1 ± 0.2


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120
µE ∙ m-2 ∙ s-1, mean value: 5878 lux,

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-Chlorophyll a-fluorescence
The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was found in the definitive test.

Reference substance (positive control):

yes

Remarks:

(Potassium dichromate)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.055 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: Calculated test item concentration using the mean measured initial concentrations of the main constituents

Basis for effect:

growth rate

Remarks on result:

other: Cl: 0.0415 - 0.0659

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.019 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

other: Calculated test item concentration using the mean measured initial concentrations of the main constituents

Basis for effect:

growth rate

Remarks on result:

other: Cl: 0.0101 - 0.0255

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

0.005 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.154 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: 0.115-0.183

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.001 mg/L

Nominal / measured:

not specified

Conc. based on:

other: calculated main constituents

Basis for effect:

other: growth rate and yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.01 mg/L

Nominal / measured:

not specified

Conc. based on:

other: calculated main constituents

Basis for effect:

other: yield

Remarks on result:

other: CI: 0.00836 - 0.0111

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.058 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI: 0.0506 - 0.0671

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.025 mg/L

Nominal / measured:

not specified

Conc. based on:

other: calculated main constituents

Basis for effect:

growth rate

Remarks on result:

other: CI: 0.0190 - 0.0302

Details on results:

The use of the mean measured initial concentration is considered justified because the substance sorbs strongly to algae and the growth rate . Due to that nigligible concentrations were observed in the aqueous phase after 72h and because sorption to glassware is observed to be nigligible. This approach is in agreement with point 40 of the OECD 201 guideline.

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: The test media clear throughout the exposure period.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

The toxicity of potassium dichromate (SIGMA ALDRICH, batch number MKBV0900V, purity 99.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2017-04-18 to 2017-04-21. The reference item toxicity is in the valid range following test facility SOPS.

EC50-Values of the Reference Item
based on nominal concentrations mg/L, (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Growth Rate inhibition
ErC50 0.559 0.754 ± 0.518
95% confidence interval 0.538 – 0.581
Yield inhibition
EyC50 0.320 0.398 ± 0.291
95% confidence interval 0.242 – 0.341
SD = Standard deviation

Reported statistics and error estimates:

EL/C-values and statistical analyses
EL/C10-, EL/C20- and EL/C50-values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression.

NOEL/NOEC, LOEL/NOEC and statistical analyses
The NOEL/NOEC and LOEL/LOEC were determined by calculation of statistically significant differences of growth rates and yield. The following statistical tests were conducted:

Shapiro-Wilk’s test on normal distribution was calculated with a significance level of 0.01.
Levene’s test on variance homogeneity was calculated with a significance level of 0.01.
Dunnett’s multiple -t-test was calculated with a significance level of 0.05 for yield.
Monotonicity of Concentration/Response was calculated by non-parametric trend analysis by contrasts (significance level 0.05) for growth rate.
SD Jonckheere-Terpsta test procedure rate was done for growth with a significance level of 0.05 for growth rate.

Calculation of initial main constituents concentrations
The recoveries for the lowest two loadings were < LOQ at test start. Therefore, and as the recoveries in all loadings > LOQ were very similar, a mean value of recovery percentages of the upper three loadings containing algae was calculated, which was 36% of the nominal values. This percentage was applied for all loadings to calculate the expected recovery. This is considered to reflect the actual concentrations in the best possible and reliable manner.

Software
The data for the tables in this report were computer-generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
•Excel, MICROSOFT CORPORATION
•SigmaPlot, SPSS INC.
•GraphPad Prism, GRAPHPAD SOFTWARE, INC.
•ToxRat Version 3.2.1, ToxRat Solutions GmbH

Cell Densities

Nominal test item loading	Calculated initial main constituents concentration	Replicate	Cell density [cells/mL]
[mg/L]	[mg m.c./L]	No.	0 hours	24 hours	48 hours	72 hours
0.200	0.0330	1	6839	7412	6052	17457
		2	6839	6994	13077	38384
		3	6839	8960	13279	55978
		Mean	6839	7789	10803	37273
0.0800	0.0132	1	6839	17288	59952	501994
		2	6839	17855	93320	817359
		3	6839	18467	94520	841147
		Mean	6839	17870	82597	720167
0.0320	0.00529	1	6839	20694	152940	1588038
		2	6839	19889	171865	1416574
		3	6839	21323	169665	1415404
		Mean	6839	20635	164823	1473339
0.0128	0.00211	1	6839	24219	199978	1871216
		2	6839	25109	207165	1680441
		3	6839	23309	199335	1713036
		Mean	6839	24212	202159	1754898
0.00513	0.000846	1	6839	22590	199873	1972924
		2	6839	24214	212619	1846169
		3	6839	26467	196514	1770035
		Mean	6839	24424	203002	1863043
Control		1	6839	27131	227565	1966779
		2	6839	28076	218904	2267338
		3	6839	25756	236858	1786947
		4	6839	26276	244220	2072993
		5	6839	28711	252295	1996800
		6	6839	25390	243236	2062927
		Mean	6839	26890	237180	2025631

Evaluation after 72 hours

   Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

Nominal test item loading	Calculated initial main constituents concentration	Replicate	Growth rate		Inhibition of growth rate	Yield		Inhibition of yield
[mg/L]	[mg m.c./L]	No.	[d^-1]		[%]	[cells/mL]		[%]
0.200	0.0330	1		0.312	84		10618	99
		2		0.575	70		31545	98
		3		0.701	63		49139	98
		Mean	(+)	0.529	72	(+)	30434	98
0.0800	0.0132	1		1.43	24		495155	75
		2		1.59	16		810520	60
		3		1.60	15		834308	59
		Mean	(+)	1.54	19	(+)	713328	65
0.0320	0.00529	1		1.82	4		1581199	22
		2		1.78	6		1409735	30
		3		1.78	6		1408565	30
		Mean	(+)	1.79	6	(+)	1466500	27
0.0128	0.00211	1		1.87	1		1864377	8
		2		1.84	3		1673602	17
		3		1.84	3		1706197	15
		Mean	(+)	1.85	3	(+)	1748059	13
0.00513	0.000846	1		1.89	0		1966085	3
		2		1.87	2		1839330	9
		3		1.85	2		1763196	13
		Mean	(-)	1.87	1	(-)	1856204	8
Control		1		1.89			1959940
		2		1.94			2260499
		3		1.86			1780108
		4		1.91			2066154
		5		1.89			1989961
		6		1.90			2056088
		Mean		1.90			2018792

 

Section-by-Section and Average Specific Growth Rates of the Control Group (0 – 72 hours)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 hours)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.38	2.13	2.16	1.89	0.441	23.4	24.5
	2	1.41	2.05	2.34	1.94	0.474	24.5
	3	1.33	2.22	2.02	1.86	0.469	25.3
	4	1.35	2.23	2.14	1.91	0.486	25.5
	5	1.44	2.17	2.07	1.89	0.400	21.1
	6	1.31	2.26	2.14	1.90	0.516	27.1
				Mean	1.90
				SD ±	0.03
				CV [%]	1.37

 

SD = Standard deviation CV = Coefficient of variation

Validity criteria fulfilled:

yes

Conclusions:

In this study, the test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values based on nominal test item loadings: The EL50-values with 95% confidence intervals for inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were 0.154 (0.115 – 0.183) mg/L and 0.0583 (0.0506 – 0.0671) mg/L, respectively. The EL10-values with 95% confidence intervals for inhibition of growth rate (ErL10) and yield (EyL10) after 72 hours were 0.0524 (0.0281 – 0.0710) and 0.0836 (< 0.00513 – 0.0161) mg/L, respectively.
The following effect values are based on the calculated initial main constituents concentrations: The EC50-values with 95% confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 0.0254 (0.0190 – 0.0302) and 0.00962 (< 0.000846 – 0.00266) mg m.c./L, respectively. The EC10-values with 95% confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 0.00863 (0.00462 – 0.0117) and 0.00138 (0.00836 – 0.0111) mg m.c./L, respectively.
The following effect values are based on the calculated initial test item concentrations which were calculated from the observed effect values for the main constituents: The EC50-values with 95% confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 0.0554 (0.0415 – 0.0659) mg m.c./L and 0.02099 (< 0.00185 – 0.00580) mg test item/L, respectively. The EC10-values with 95% confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 0.0188 (0.0101 – 0.0255) and 0.00301 (0.00182 – 0.0242) mg test item/L, respectively.

Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016 C.3 from 2017-04-24 to 2017 -06-26 with the definitive exposure phase from 2017-06-20 to 2017-06-23, at the test facility. The aim of the study was the determination of NOEL, LOEL, EL₁₀, EL₂₀- and EL₅₀-values of growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 6839 cells/mL. As the test item is poorly water soluble and an UVCB substance, five water accommodated fractions (WAF) with the nominal test item loadings 0.00513 -0.0128 – 0.0320 - 0.0800 - 0.200 mg/L (factor 2.5), corresponding to the calculated initial main constituents concentrations 0.000846 – 0.00211 – 0.00529 – 0.0132 – 0.0330 mg m.c./L were prepared with dilution water. For the loading of 0.500 mg/L an appropriate amount of the test item was weighed out. The test item was applied onto a glass slide. The glass slide with the test item was inserted into a brown glass flask with an appropriate amount of dilution water. The dispersion was shaken for at least 24 hours with 20 rpm at room temperature. After a separation phase of 1 hour, the WAF was removed by siphoning (from the approximate centre of the glass flask). The resulting WAF was used as a stock solution and all tested loading levels were prepared by diluting the WAF, since the concentrations were too low for direct weighing.Three replicates were tested for each test item loading and six replicates for the control. The environmental conditions were within the acceptable limits.

 

The test media were clear throughout the test period. The concentrations of the test item Redicote 814M in all test concentration levels with and without algae and the control were analytically verified by LC-MS/MS at the start and at the end of exposure. Additionally, the adsorption to glass was analytically verified from all concentration levels with and without algae.

 

The measured concentrations of the test item in the fresh media (0 h) with and without algae were between < LOQ and 55% of the nominal concentrations for the three main constituents C16 diamine, C18:1 diamine and C18:0 diamine. The measured concentrations in the media after 72 hours with and without algae were between < LOQ and 7% of the nominal concentrations for the three main constituents C16 diamine, C18:1 diamine and C18:0 diamine. The measured concentrations of the adsorption to glass at the end of the exposure with and without algae were between < LOQ and 13% of the nominal values for the component C16 diamine, < LOQ for the component C18:1 diamine and < LOQ and 17% for the component C18:0 diamine.

 

Therefore, all effect values given are based on the nominal test item loadings and the calculated initial main constituents concentration of the test item. In addition were the main constituent concentrations recalculated to the test item concentrations using the fraction of different constituents in the test item.

 

NOEL/NOEC, LOEL/LOEC, EL_x/EC_x-values and 95% Confidence Intervals of Redicote 814M (0 - 72 hours)

                  based on nominal test item loadings and the calculated initial main constituents and calculated test item concentrations [mg/L]

	Growth Rate Inhibition
	Nominal test item loading [mg/L]	Calculated main constituents [mg m.c./L]	Calculated test item [mg./L]
NOEL/NOEC	0.00513	0.000846	0.00185
LOEL/LOEC	0.0128	0.00211	0.00460
ErL10/ ErC10	0.0524 (0.0281 – 0.0710)	0.00863 (0.00462 – 0.0117)	0.0188 (0.0101 – 0.0255)
ErL20/ ErC20	0.0834 (0.0665 – 0.139)	0.0138 (0.0110 – 0.0230)	0.0301 (0.0240 – 0.0502)
ErL50/ ErC50	0.154 (0.115 – 0.183)	0.0254 (0.0190 – 0.0302)	0.0554 (0.0415 – 0.0659)
	Yield Inhibition
	Nominal test item loading [mg/L]	Calculated main constituents [mg m.c./L]	Calculated test item [mg/L]
NOEL/NOEC	0.00513	0.000846	0.00185
LOEL/LOEC	0.0128	0.00211	0.00460
ErL10/ ErC10	0.00836 (< 0.00513 – 0.0161)	0.00138 (0.000836 – 0.0111)	0.00301 (0.00182 – 0.0242)
ErL20/ ErC20	0.0229 (0.0168 – 0.0304)	0.00379 (0.00278 – 0.00503)	0.00827 (0.00607 – 0.01100)
ErL50/ ErC50	0.0583 (0.0506 – 0.0671)	0.00962 (< 0.000846 – 0.00266)	0.02099 (0.00185 – 0.00580)

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2008-02-04 to 2008-02-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Complete report, test performed under GLP, all validity criteria fulfilled

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Melting point: 9-20 °C
- Boiling point: > 250 °C
- Vapour pressure: < 1 hPa (20 °C)
- Water solubility (under test conditions): Insoluble
- Henry's law constant: Not applicable
- log Pow: Not applicable
- pKa: Not applicable
- Stability in water: Not applicable
- Stability in light: Not applicable
- pH dependance on stability: Not applicable

OTHER PROPERTIES (if relevant for this endpoint)
- Results of test for ready biodegradability: Not applicable
- Other:
- Density: 840 kg/m3 (25 °C)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 10.0 - 32.0 - 100 - 320 - 1000 µg/L
- Sampling method: Each sample was diluted 1:2 with 2-propanol containing 4 % formic acid directly after sampling for stabilisation
- Sample storage conditions before analysis: Room temperature

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 20 mg/L, freshly prepared with dilution water 30 min before application, sonificated for 30 min at 40 °C
- Differential loading: 10-32-100-320-1000 µg/L (factor: √10)
- Controls: six replicates without test item, tested under the same conditions as the test replicates
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): None
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Not observed

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG, Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted 35-70 µE•m-2•s-1 for 24 h per day

ACCLIMATION
- Culturing media and conditions (same as test or not): Inoculum: Nutrient medium Z according to Luettge (1994), conditions same as for test; test replicates: natural river water, other conditions same as for test
- Any deformed or abnormal cells observed: No

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

After 72 h algae were transferred from nominal concentrations 320 – 1000 µg/L and control into fresh untreated medium (0.5 – 1 mL algae suspension from each replicate were filled up with dilution water to 10 mL) and allowed to grow for further 5 d to determine whether the effect of the item is reversible.

Hardness:

Not measured

Test temperature:

Mean: 21.5 °C, Min: 21 °C, Max: 22 °C

pH:

Nominal test item concentration pH-value
[µg/L] Start; 0 h End; 72 h
1000 7.76 7.70
320 7.77 7.66
100 7.73 7.87
32 7.76 8.04
10 7.86 7.76
Control 7.92 8.03

Dissolved oxygen:

Not measured

Salinity:

Not measured

Nominal and measured concentrations:

Nominal test item concentrations: 10-32-100-320-1000 µg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): None
- Initial cells density: 4971 cells/mL
- Control end cells density: Mean 774306 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Natural river water, river grane, located in the low mountain range "Harz", D-38685 Langelsheim, Herzog-Julius-Hütte, Im Granetal; additionally 50 % of the components of the dilution water acc. to the guideline were added to enable a sufficient growth of algae
- Particulate matter: 2.5 mg/L
- DOC 1.5 mg C/L
- Conductivity: 149 µS (natural river water)
- Salinity: Not measured
- Culture medium different from test medium: Nutrient medium Z according to LÜTTGE et al. (1994)

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: No
- Photoperiod: 24 h
- Light intensity and quality: 60-120 µE x m-2 x s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Microscopic
- Chlorophyll measurement: fluorimeter; Chlorophyll-a-fluorescence, excitation at 435 nm, emission at 685 nm; measurements at test start and every 24 h, each replicate was measured 6-fold, value (flash) no. 3 - 6 were used for calculations
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations: √10
- Range finding study
- Test concentrations: 10-100-1000 µg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

507 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 468 - 543

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

188 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 167-206

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: none
- Colour differences: none
- Flocculation: None
- Adherence to test vessels: None
- Aggregation of algal cells: at test end of the concentration levels 320 - 1000 µg/L
- Other: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
- Effect concentrations exceeding solubility of substance in test medium: None

The study meets the validity criteria of the guideline:
- The cell growth increased 156-fold after 72 h in the control (required: 16-fold).
- The temperature during the test was in the range of 21-24 °C. The difference from the mean was within ± 2 °C: min.: 21 °C, max.: 22 °C mean value: 21.5 °C.
- The pH-value in the control replicates increased not higher than 1.5 units: from 7.92 to 8.03.
- The mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures was 27.71 % (required: < 35 %).
- Coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 1.94 % (required: < 7 %).

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 1.01 (CI 0.90 - 1.13) mg/L
Yield inhibition: 0.42 (CI 0.37 - 0.48) mg/L

Reported statistics and error estimates:

EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 h were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals EC10-, EC20- and EC50-values were carried out using maximum likelihood analyses.

Table 1: EC₁₀-_, EC₂₀- and EC₅₀- values (0-72 h) of N-Oleyl-1,3-diaminopropane

                based on nominal test item concentrations [µg/L]

                            

	Inhibition of yield
EyC10 (95 % confidence interval)	113 (98 – 131)
EyC20 (95 % confidence interval)	137 (121 – 158)
EyC50 (95 % confidence interval)	214 (186 – 243)
	Rate-related inhibition
ErC10 (95 % confidence interval)	188 (167 – 206)
ErC20 (95 % confidence interval)	267 (244 - 291)
ErC50 (95 % confidence interval)	507 (468 – 543)

Table 2:     Cell Densities

	Nominal test item concentration		Replicate		Cell density [cells/mL]
[µg/L]		No.		0 h		24 h	48 h	72 h
1000		1		4971		10547	7050	12720
		2		4971		9255	8373	13602
		3		4971		11649	9948	9255
		Mean		4971		10484	8457	11859
320		1		4971		38393	108669	207390
		2		4971		33920	100605	159573
		3		4971		39653	142595	230732
		Mean		4971		37322	117290	199232
100		1		4971		42519	223266	667416
		2		4971		43275	210351	685056
		3		4971		41291	215076	808536
		Mean		4971		42362	216231	720336
32		1		4971		41984	239961	892326
		2		4971		41417	260751	868071
		3		4971		45858	219171	848856
		Mean		4971		43086	239961	869751
10		1		4971		39716	237126	789006
		2		4971		44945	288156	777981
		3		4971		42992	198066	896421
		Mean		4971		42551	241116	821136
Control		1		4971		40661	240276	730101
		2		4971		44756	225471	674346
		3		4971		42897	241536	798456
		4		4971		38298	218226	794361
		5		4971		41921	201216	748056
		6		4971		44409	252246	900516
		Mean		4971		42157	229829	774306

Table 3     Evaluation after 72 h

        

Nominal test item concentration	Replicate	Yield		Inhibition of yield	Growth rate		Rate-related inhibition
[µg/L]	No.			[%]	[d^-1]		[%]
1000	1		7749	98.99		0.313	81.37
	2		8631	98.88		0.336	80.04
	3		4284	99.44		0.207	87.68
	Mean		6888	99.10		0.285	83.03
320	1		202419	73.69		1.244	26.04
	2		154602	79.90		1.156	31.23
	3		225761	70.66		1.279	23.92
	Mean		194261	74.75		1.226	27.06
100	1		662445	13.89		1.633	2.86
	2		680085	11.60		1.642	2.35
	3		803565	-4.45		1.697	-0.94
	Mean		715365	7.01		1.657	1.42
32	1		887355	-15.34		1.730	-2.89
	2		863100	-12.19		1.721	-2.35
	3		843885	-9.69		1.713	-1.90
	Mean		864780	-12.41		1.721	-2.38
10	1		784035	-1.91		1.689	-0.45
	2		773010	-0.48		1.684	-0.17
	3		891450	-15.87		1.732	-2.98
	Mean		816165	-6.09		1.702	-1.20
Control	1		725130			1.663
	2		669375			1.637
	3		793485			1.693
	4		789390			1.691
	5		743085			1.671
	6		895545			1.733
	Mean		769335			1.681

Negative values = increase of growth

Table 4:     Section-by-Section and Average Specific Growth Rates of the Control Group
               (0 – 72 h)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	VC %	Mean VC %
		section-by-section
		0 - 24 h	24 - 48 h	48 - 72 h
Control	1	2.102	1.777	1.111	1.663	0.505	30.35	27.71
	2	2.198	1.617	1.096	1.637	0.551	33.68
	3	2.155	1.728	1.196	1.693	0.481	28.39
	4	2.042	1.740	1.292	1.691	0.377	22.31
	5	2.132	1.569	1.313	1.671	0.419	25.08
	6	2.190	1.737	1.273	1.733	0.459	26.46
				Mean	1.681
				SD ±	0.03
				VC %	1.94

SD = Standard deviation        VC %= percent coefficient of variation

Table 5:     Recovery of Alga Cells

Nominal test item concentration                   [µg/L]	Incubation time                    [d]	Cell density [cells/mL]		Growth rate after incubation time [d^-1]
		Initial^#	Final
1000	5	1186	72255	0.82
			76917	0.83
			54867	0.77
320		19923	242229	0.50
			203295	0.46
			217407	0.48
Control		38715	201909	0.33
			250041	0.37

^#) calculated from the cell density of the definitive test after 72 h

Validity criteria fulfilled:

yes

Remarks:

The study meets the validity criteria of the guideline

Conclusions:

The test is considered valid as all validity criteria have been fulfilled; Based on the test setup (GLP, Guideline study) and the extensive effort which has been paid to ascertain a realistic exposure of the test organisms and to quantify the extent of this exposure the results are considered to be reliable. Finally, the test result is considered adequate for the evalution of the environmental risks in the EU applying the bulk approach considering the realistic worst case (low) concentration of suspended matter (2.5 mg/L) and DOC (1.5 mg C/L) in the river water used.

Executive summary:

The toxicity of N-Oleyl-1,3-diaminopropane (CAS No. 7173-62-8) (Batch no.: S000902)to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorien in 31157 Sarstedt, Germany from February 04 to 12, 2008. The aim of the study was to assess the effects on growth rate and yield over a period of 72 h.

An individual test design was applied. Natural river water was used as dilution medium.

The study was conducted under static conditions with an initial cell density of nominal 2 to 5 x 10³cells/mL. Five concentrations were tested in a geometrical series with a dilution factor of √10, nominal: 10 - 32 - 100 - 320 - 1000 µg/L. Three replicates were tested for the test item concentrations and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of C18 : 1 diamine, main component of The test substance were analysed at all concentration levels at test start and test end via LC-MS/MS analysis. The measured concentrations at test start were in the range of 91 to 98 % of the nominal values. At the end of the test N-Oleyl-1,3-diaminopropane gave recoveries below the limit of quantification up to 12 % of the nominal values. Biodegradation as possible reason for this decrease is very unlikely considering the short time frame, low DOC and suspended matter content and also because the river water was frozen before use to minimize the microbial activity. This is supported by the monitoring results from the chronic daphnia tests with this test item. In this test only a limited decrease of the test substance was observed over 48 hours. This decrease was attributed to additional sorption to suspended matter and DOC due to thermodynamically driven redistribution of the sorbed fraction. The larger decrease of the test item during the algae test is therefore most likely caused by sorption of the test substance to algae. These points support the conclusion that the test organisms were fully exposed to the test substance during the test. At the end of the test N-Oleyl-1,3-diaminopropane gave recoveries ranging from LOQ (2.04 µg/L) to 12 % of the nominal values. Therefore all effect values are given based on nominal concentrations of the test item. In this study N-Oleyl-1,3-diaminopropane was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h with the following effect values (nominal concentrations): The EC₁₀-values with 95 % confidence intervals for inhibition of yield (E_yC₁₀) and specific growth rate (E_rC₁₀) after 72 h were 113 (98 to 131) and 188 (167 to 206) µg/L, respectively. The EC₅₀-values with 95 % confidence intervals for inhibition of yield (E_yC₅₀) and specific growth rate (E_rC₅₀) after 72 h were 214 (186 to 243) and 507 (468 to 543) µg/L, respectively.

After 72 h algae were transferred from the nominal concentrations 320 to 1000 µg/L and control to fresh untreated medium and allowed to grow for further 5 d under test conditions. The test item effect was observed to be reversible at these concentrations. Therefore, there is potential for recovery following exposure up to 1000 µg/L (highest test concentration)

Description of key information

One toxicity test with the green alga Pseudokirchneriella subcapitata according to OECD 201 has been performed with"Reaction mass of Amines, N-tallow alkyltrimethylenedi-, (2-ethylhexanoates), Amines, N-tallow alkyltrimethylenedi-, acetates and n-tallow-1,3 -diaminopropane ditallate" (R814M). This substance is the salt of tallow-1,3 -diaminopropane (CAS no 1219010 -04 -4) and a mixture of talloil fatty acids, 2 -ethylhexoic and acetic acid. The 72h ErC50 and ErC10 based on main constituents is respectively 0.0254 (95% CL: 0.0190 – 0.0302) and 0.00863 (95% CL: 0.00462 – 0.0117 mg m.c./L.

Finally these results were recalculated to the effect data for the test item resulting in a 72h ErC50 and ErC10 of respectively 0.0554 (95% CL: 0.0430 – 0.0511) and 0.0188 (95% CL: 0.0101 – 0.0255) mg test item/L.

Under aquatic environmental conditions a large extend of the salt is expected to be dissociated and ecotoxicity observed will originate mainly from tallow-1,3 -diaminopropane. No effects (NOEL>=100 mg/L) were observed for the WAF of tall oil fatty acid to algae and daphnids and for 2 -ethyl hexanoic acid the lowest NOEC (21d daphnia) was 25 mg/L (ECHA). Therefore available ecotoxicity data on alkyl-1,3-diamines were included in the dataset. The ecotoxicity of tallow-1,3 -diaminopropane is expected to be reduced when forming a salt with talloil fatty acids or 2 -ethylhexoic acid.

One study with oleyl-1,3 -diaminopropane which was performed in river water which resulted in an ErC10 and ErC50 of respectively 0.188 and 0.507 mg/L.

Recalculating these results to R814M using the tallow-1,3 -diaminopropane content of 52.2% in R814M gives a ErC10 and ErC50 of respectively 0.360 and 0.97 mg/L. These ErC values of 0.360 and 0.97 mg/L will be as a worst-case be used as Key value for risk assessment.

The result as observed in the algae study of 0.0554 mg/L with R814M can be used as Key study for C&L.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.97 mg/L

EC10 or NOEC for freshwater algae:

360 µg/L

Additional information

One toxicity test with the green alga Pseudokirchneriella subcapitata according to OECD 201 has been performed with"Reaction mass of Amines, N-tallow alkyltrimethylenedi-, (2-ethylhexanoates), Amines, N-tallow alkyltrimethylenedi-, acetates and n-tallow-1,3 -diaminopropane ditallate" (R814M). This substance is the salt of tallow-1,3 -diaminopropane (CAS no 1219010 -04 -4) and a mixture of talloil fatty acids, 2 -ethylhexoic and acetic acid.

Due to the complex structure of the test item, the WAF approach was used. The study was conducted under static conditions over a period of 72 hours using a stock solution which was prepared as a WAF (0.5 mg/L) of the test item. The test solutions were prepared by diluting the WAF to the following nominal concentration range : 0.00513 -0.0128 – 0.0320 - 0.0800 - 0.200 mg/L (factor 2.5). The concentrations of the main amine constituents (diamine C18:1, diamine C16 and diamine C18) of the test item were analytically verified via LC-MS/MS in fresh media at the start of the exposure and in the old media (72h) in all concentration levels and the control. Additionally, the adsorption to glass was analytically verified from all concentration levels with and without algae. The chemical analysis was focused on the amine fraction in the test solutions because this fraction is considered to be the most toxic fraction in the test item. The measured concentrations of the three main constituents (m.c.) of the amine fraction of the test item did not remain within ± 20% of the nominal concentrations and therefore the mean measured initial concentrations of the sum of the three main constituents was calculated and are reported in addition to the nominal loading levels. The use of the mean measured initial concentration is considered justified because of the strong sorption of the test item to the algae (see OECD 201 point 40). The observed mean measured initial concentrations of the main constituents were: 0.000846 – 0.00211 – 0.00529 – 0.0132 – 0.0330 mg m.c./L

The dose effect data derived using the nominal loading concentrations resulted in a 72h ErL50 and ErL10 of respectively 0.154 (95% CL: 0.115 – 0.183) and 0.0524 (95% CL: 0.0281 – 0.0710) mg/L.

The 72h ErC50 and ErC50 based on main constituents is respectively 0.0254 (95% CL: 0.0190 – 0.0302) and 0.00863 (95% CL: 0.00462 – 0.0117 mg m.c./L

Finally these results were recalculated to the effect data for the test item resulting in a 72h ErC50 and ErC10 of respectively 0.0554 (95% CL: 0.0430 – 0.0511) and 0.0188 (95% CL: 0.0101 – 0.0255) mg test item/L.

Under environmental conditions a large extend of the salt is expected to be dissociated and ecotoxicity observed will originate mainly from tallow-1,3 -diaminopropane. Therefore available ecotoxicity data on alkyl-1,3-diamines were included in the dataset. The ecotoxicity of tallow-1,3 -diaminopropane is expected to be reduced when forming a salt with talloil fatty acids or 2 -ethylhexoic acidbutbecause of the availability of real measured acute toxicity data for the test substance (R814M). The availabledataon alkyl-1,3 -diaminopropanes was included for comparison reasons.

For alkyl-1,3 -diaminopropanes two studies with algae are available. One older study from 2000 where a 72h EC50 of 0.046 mg/L for tallow-1,3 -diaminopropane was observed using lab water with a reliability score of 2 e.g. because the exposure concentrations were not quantified and the mean coefficient of variation for section-by-section specific growth rates in the control cultures exceeded 35%. One study with oleyl-1,3 -diaminopropane which was performed in river water which resulted in an ErC10 and ErC50 of respectively 0.188 and 0.507 mg/L.

Recalculating these results to R814M using the tallow-1,3 -diaminopropane content of 52.2% in R814M gives a ErC10 and ErC50 of respectively 0.360 and 0.97 mg/L. These ErC values of 0.360 and 0.97 mg/L will be as a worst-case be used as Key value for risk assessment.

The result as observed in the algae study of 0.0554 mg/L with R814M can be used as Key study for C&L.

There is also an algae study (OECD 201, river water, reliability 1) available with tallow-1,3 -diaminopropane oleate (CAS no 1307863 -78 -0) which can be considered to be a comparable substance to the talloil fatty acid salt of tallow-1,3 -diaminopropane. This study resulted in a 72h ErC10 and ErC50 of respectively 0.324 and 0.509 mg/L.